Device with chemical surface patterns

ABSTRACT

A device with chemical surface patterns (defined surface areas of at least two different chemical compositions) with biochemical or biological relevance on substrates with prefabricated patterns of at least two different types of regions (α, β, . . . ), whereas at least two different, consecutively applied molecular self-assembly systems (A, B, . . . ) are used in a way that at least one of the applied assembly systems (A or B or . . . ) is specific to one type of the prefabricated patterns (α or β or . . . ).

BACKGROUND OF THE INVENTION

The invention relates to a device with chemical surface patterns, abioanalytical sensing platform comprising the device, a method for thesimultaneous determination of analytes and a biomedical device.

Chemical patterning of surfaces i.e., the generation of structures ofdifferent chemical composition on surfaces, either in a regular,geometric array or with a statistical distribution of features, is animportant technique in a variety of application includingmicrofabrication, microelectronics, micromechanics, biomaterials andbiosensors [Kane, R. S., Takayama, S., Ostuni, E., Ingber, D. E.,Whitesides, G. M., Patterning proteins and cells using soft lithography,Biomaterials 20 (1999) 2363-2376. Xia, Y., Rogers, J. A., Paul, K. E.,Whitesides, G. M., Unconventional Methods for Fabricating and PatterningNanostructures. Chem. Rev. 99 (1999), 1823-1848]. FIG. 1 shows examplesof chemical patterns, which may or may not be connected withtopographical variations (height differences) across the surface.

A large variety of techniques has been developed and described in theliterature and in patents to produce patterns with more or lesscontrolled chemical composition and structure in different areas of asurface. Examples include:

Type A: Techniques that involve the use of photoresists and/or etchingprocedures [Xia, Y., Rogers, J. A., Paul, K. E., Whitesides, G. M.,Unconventional Methods for Fabricating and Patterning Nanostructures.Chem. Rev. 99 (1999), 1823-1848]

-   -   Lithography using visible, UV or X-ray exposure of        photosensitive coatings (photoresists) through appropriate masks    -   Electron beam lithography    -   Writing structures by fast ion bombardement    -   Laser microstructuring    -   And many other techniques

Type B: Techniques that rely on self-assembly: A number of techniquesuse molecular self-assembly in combination with structuring techniques:

-   -   Microfluidic patterning (μFP) of surfaces in contact with stamps        having channels that can be filled with a solution containing        molecules that assemble on the exposed surface within the        channels [Kane, R. S., Takayama, S., Ostuni, E., Ingber, D. E.,        Whitesides, G. M., Patterning proteins and cells using soft        lithography, Biomaterials 20 (1999) 2363-2376].    -   Microcontact printing (μCP), where stamps with a particular        structure are used to transfer material locally adsorbed at or        absorbed in the stamp to the surface in a selective way        [Kane, R. S., Takayama, S., Ostuni, E., Ingber, D. E.,        Whitesides, G. M., Patterning proteins and cells using soft        lithography, Biomaterials 20 (1999) 2363-2376. Chiu, D. T.,        Jeon, N. L., Huang, S., Kane, R., Wargo, C. J., Choi, I. S.,        Ingber, D. E., Whitesides, G. M., Patterned deposition of cells        and proteins onto surfaces by using three-dimensional        microfluidic systems. Proc. Natl. Ac. Sci. 97 (2000),        2408-2413].    -   UV patterning of alkane thiols on gold surfaces through        appropriate masks, resulting in spatially controlled oxidation        of the thiol headgroup to sulfuroxide or sulfon, washing off the        surface the less strongly bound oxidized alkane thiols and        backfilling the unprotected gold areas with a different thiol        [Xia Y N, Zhao X M, Whitesides G M, Pattern transfer:        Self-assembled monolayers as ultrathin resists, Microelectronic        Engineering 32 (1-4): 255-268, September 1996].

These standard techniques described above have, however, specificdisadvantages:

Type A techniques, although partly suitable for mass scale production,in general only allow the fabrication of structures with relativelysimple surface chemistries, meaning chemical compositions that have tobe stable in the development stages of the lithographic process. In thebiomaterial and biosensor area, however, there is a requirement tostructure surfaces based on rather delicate, often labile molecules suchas proteins, antibodies or nucleic acids (DNA or RNA). The harshconditions of the lithographic fabrication steps are likely to beincompatible with these types of biochemical or biological structures.

Type B: While these techniques allow the spatially-controlled transferof highly sensitive molecules such as proteins, they always involve alocal contact of the surface with the stamping material, which may leadto the transfer of unwanted stamp material and thus local contaminationthat may interfere with the functionality of the surface. The standardstamp materials are based on elastomeric siloxane or silicon type ofpolymers [Patent number: WO 9629629, publication date: Sep. 26, 1996,inventor(s): Jackman Rebecca J, Whitesides George M; Biebuyck Hans; KimEnoch; Mrksich Milan; Berggren Karl K; Gorman Chris; Kumar Amit;Prentiss Mara G; Wilbur James L; Xia Younan, Applicant(s): HarvardCollege (US)], such as polydimethylsiloxane, and these are particularlycritical in terms of transfer of low-molecular-weight or monomericcomponents of the stamp elastomer to surfaces, leading to hydrophobiccontaminated contact surfaces, which are likely to interfere withsubsequent modification procedures. Another major disadvantage is thelack of reproducibility due to variations in quality from stamp tostamp, and the general difficulty of patterning large areas due todifficulties of achieving a perfect stamp-surface contact area overlarger dimensions. Moreover, there are restrictions in the type ofpatterns that can be produced by stamping using elastomer stamps; e.g.widely-spaced patterns can not be transferred efficiently due to thesagging of the stamp. Finally, when using stamps in production, there isa continuous deterioration in the fidelity of the stamping process overthe life time of a stamp.

The invention aims at eliminating some of the major disadvantages andlimitations of the known techniques described in the introduction.Firstly, it aims at providing a patterning technology that allows one topattern large surfaces and large batch sizes in a very reproducible way,with almost no limitations in terms of the geometry and dimensions ofthe patterns. Secondly, it provides the ability to fabricate patternswith biochemical or biological structures such as peptides, proteins, ornucleci acids. A particularly important aim is to pattern surfaces intoareas that are resistant to interactions with biological media, meaning,in particular, resistance to the adsorption of biomolecules (e.g.proteins, carbohydrates or nucleic acids) and cells, and areas thatelicit specific biological responses, such as antibody-antigeninteractions or cell receptor-surface interactions.

There are a number of particular needs that the invented technique isable to address:

-   -   Flexibility-basically without limitations-with respect to the        geometry and size of the features, ranging from the mm across        the micrometer to the submicrometer and nanometer range.    -   Stringent control over the physico-chemical properties of the        pattern areas.    -   Extremely high contrast between adhesive and non-adhesive areas,        meaning very high ratios of protein adsorption or cell        attachment on the adhesive area in relation to the non-adhesive        “background”.    -   The possibility that the biochemical or biological modification        is directly linked to the selective adsorption in areas of        defined physico-chemical properties.    -   Stringent control over the density, conformation, orientation        and therefore functionality of biochemically or biologically        active sites immobilized in specific areas of the pattern.    -   High reproducibility and fidelity of the pattern chemistry and        biology across large surfaces areas, and with little or no        variations from batch to batch.

SUMMARY OF THE INVENTION

The objectives are met by a device with a chemical surface patterns(defined surface areas of at least two different chemical compositions)with biochemical or biological relevance on substrates withprefabricated patterns (“prepatterns”) of at least two different typesof regions (called α, β, . . . ), whereas at least two different,consecutively applied molecular self-assembly systems (called A, B, . .. ) are used in a way that at least one of the applied assembly systems(A or B or . . . ) is specific to one type of the prefabricated patterns(α or β or . . . ) (see schematic process scheme in FIG. 2).

A preferred example is a device where the specificity is achievedthrough self-assembly of alkane phosphates or alkane phosphonates fromaqueous solutions (assembly system A) in combination with prepatternedsurfaces whereas only one type of the prepattern area (a) forms amolecularly assembled layer A of alkane phosphates, while the otherprepattern area(s) (β, . . . ) remains uncoated (“selective chemicalreactivity contrast”).

Another preferred example is a device of where α is an oxide, nitride orcarbide of a metal that chemically interacts with phosphates and/orphosphonates, in particular transition metal oxides such as titaniumoxide, tantalum oxide, niobium oxide, zirconium oxide, or non-transitionmetal oxides that chemically interact with phosphates or phosphonates,and where b is an oxide that does not interact, in particular siliconoxide.

Another preferred example is s device of where the specificity isachieved through assembly of polyionic, PEG-grafted polymers (B) fromaqueous solution at a pH chosen such that one of the two or moreprepattern areas (e.g. β) is charged oppositely in comparison to thepolyionic copolymer and becomes coated by the copolymer due toelectrostatic interactions, while the other prepattern area(s) (e.g. α)at the same pH carries a charge of same sign as the copolymer and doesnot or does less become coated (“electrostatic contrast”).

Another preferred example is a device where the prepattern area β is anoxide, nitride or carbide with an isoelectric point (IEP) that is lowerthan that of area α and the assembly system is a (at the pH ofapplication) polycationic copolymer and the pH of the assembly systemsolution is chosen between the IEP of area α and area β.

Another preferred example is a device where the prepattern area β is anoxide, nitride or carbide with an isoelectric point (IEP) that is higherthan that of area α and the assembly system is a (at the pH ofapplication) polyanionic copolymer and the pH of the assembly systemsolution is chosen between the IEP of area α and area β.

Another preferred example is a device where the specificity is achievedthrough self-assembly of a di- or multiblock copolymer with hydrophobicand hydrophilic segments interacting with a substrate where one of theprepattern area (α) is more hydrophobic than the remaining areas, andtherefore gets coated by the di- or multiblock copolymer A(“hydrophobic-hydrophilic contrast”) while the other prepattern area (β)remains uncoated or less coated (schematic process scheme in FIG. 9).

Another preferred example is a device where the di- or multiblockcopolymer is a polypropylene oxide (PPO)-poly(ethylene glycol) (PEG)copolymer imparting protein resistance to the more hydrophobic surface.

Another preferred example is a device where the hydrophobic prepatternarea (α) is composed of a hydrophobic polymer or of an oxide that hasbeen hydrophobized through silanization or application of an alkanephosphate self-assembly system, while the hydrophilic prepattern area iseither composed of a hydrophilic polymer or is an inherently hydrophilicoxide or is an oxide that has been made permanently hydrophilic throughapplication of a self-assembled monolayer using a molecule withhydrophilic terminal functional group.

Another preferred example is a device where in a second molecularassembly step B the prepattern area β that has not been coated with thealkane phosphate becomes coated with a protein-resistant polymericlayer, leading to a final pattern that is interactive with a biologicalenvironment (proteins, cells) in areas A and not interactive (“protein-and cell-resistant”) in areas B.

Another preferred example is a device where B is the assembly of apolyionic PEG coated copolymer, adsorbing onto the oppositely chargedarea β, e.g. polycationic poly(L-lysine)-g-poly(ethylene oxide)adsorbing at pH of between 2 and 8 onto negatively charged siliconoxide.

Another preferred example is a device where in a second step theprepattern area α becomes coated with a functionalized polyionicPEG-grafted copolymer A through application of the second self-assemblysolution at a pH different from step 1, at which pH the area α is nowoppositely charged in comparison to the polyionic copolymer A andbecomes coated with the functionalized polymer, leading to a finalpattern that is interactive with a biological environment (proteins,cells) in areas A and non-interactive (“protein- and cell-resistant”) inareas B.

Another preferred example is a device where the polyionic PEG-gratedcopolymer is functionalized at the end of the PEG chains throughcovalent linkage to a biologically active group such as biotininteracting with streptavidin, or a peptide or a protein, interactingspecifically with receptors in cell membranes.

Another preferred example is a device where in a second step the morehydrophilic area (β) that has not been coated in assembly step A getscoated in the second assembly step B with a molecule that inducesspecific or non-specific interaction with the biological environment.

Another preferred example is a device where B is a functionalizedpolyionic PEG-grafted copolymer that interacts electrostatically withthe oppositely charged surface β or is an alkane phosphate that turnsthe area β into a hydrophobic, non-specifically interactive arearesulting in a final interactive/non-interactive pattern.

Another preferred example is a device where a oligo(ethylene oxide)functionalized alkane phosphate is used as the molecular assembly systemA, leading to a non-interactive area A, while the area β aresubsequently treated with an assembly system B that renders this areainteractive, e.g. by adsorbing a functionalized, polyionic PEG-graftedco-polymer.

Another preferred example is a device where a functionalized (e.g.biotin or peptide or reactive chemical group attached at end of PEGchains) PPO-PEG diblock or PEG-PPO-PEG triblock, or multiblock copolymeris used to render the correspondingly covered area specificallyinteractive, followed by a second assembly system that renders theremaining area non-interactive, e.g. through adsorption of a polyionicPEG-coated copolymer.

Another preferred example is a device where after application ofassembly system A and B the resulting interactive/non-interactivepattern is further modified through selective treatment of area A and/orB with biochemically or biologically relevant molecules.

Another preferred example is a device where the selective treatment is anonspecific adsorption of proteins or other biomolecules to the areathat is (non-specifically) interactive, e.g. hydrophobic or a selectiveinteraction with ligands previously immobilized in step A or B, e.g.streptavidin interacting specifically with biotin ligand on one of thepattern area.

Another preferred example is a device where living cells are added topatterned surfaces and become immobilized selectively on one of thepattern area, through interaction with selectively and nonspecificallyadsorbed protein or proteins, or through specific interactions withbioligands such as peptides or proteins that have in a previous stepbeen immobilized through covalent attachment to one of the patternareas.

Another, preferred subject of the invention is a bioanalytical sensingplatform comprising a “device with chemical surface pattern” accordingto any of the embodiments disclosed above and at least one biological orbiochemical or synthetic recognition element, for the specificrecognition and/or binding of one or more analytes and/or for thespecific interaction with said analyte(s), immobilized either directlyor mediated by a self-assembled layer and/or by an adhesion-promotinglayer on at least one of the different types of regions a or b or . . ..

It is preferred that the biological or biochemical or syntheticrecognition element is attached to at least one of the appliedself-assembly systems A or B, or adsorbs on at least one of saidself-assembly systems.

It is further preferred that the biological or biochemical or syntheticrecognition elements are immobilized in a one-or two-dimensional arrayof discrete measurement areas, wherein a single discrete measurementarea is defined by the area occupied by said immobilized biological orbiochemical or synthetic recognition elements on an individual, closedregion a or b.

Up to 1,000,000 measurement areas can be provided in a two-dimensionalarrangement on one “device with chemical surface pattern”, and a singlemeasurement area can occupy an area between 10⁻⁴ mm² and 10 mm².

It is preferred that the measurement areas are arranged at a density ofat least 10, preferably of at least 100, most preferably of at least1000 measurement areas per square centimeter.

The biological or biochemical or synthetic recognition elements can beselected from the group comprising proteins, such as mono- or polyclonalantibodies or antibody fragments, peptides, enzymes, aptamers, syntheticpeptide structures, glycopeptides, oligosaccharides, lectins, antigensfor antibodies (e.g. biotin for streptavidin), proteins functionalizedwith additional binding sites (“tag proteins”, such as “histidin-tagproteins”), nucleic acids (such as DNA, RNA, oligonuelotides orpolynucleotides) and nucleic acid analogues (such as peptide nucleicacids, PNA) or their derivatives with artificial bases, soluble,membrane-bound proteins, such as membrane-bound receptors and theirligands. Also whole cells or cell fragments can be immobilized forspecific recognition and detection of one or more analytes.

It is preferred, that whole cells or cell fragments, for determinationof different analytes, are immobilized in discrete measurement areas.

It is desired to minimize the amount of biological material required forthe detection of a certain analyte. The amount of necessary material isdependent on the sensitivity of the detection step. It is desired thatless than 100, preferably less than 10, most preferably only 1-3 cellsor cell fragments be immobilized per measurement area.

Many embodiments of a bioanalytical sensing platform according to theinvention are characterized in that said sensing platform is operapablefor analyte determination by means of a label, which is selected fromthe group comprising luminescence labels, especially luminescentintercalators or “molecular beacons”, absorption labels, mass labels,especially metal colloids or plastic beads, spin labels, such as ESR andNMR labels, and radioactive labels.

On the other side, refractive methods do not necessarily require the useof a label. In this context, methods for generation of surface plasmonresonance in a thin metal layer on a dielectric layer of lowerrefractive index can be included in the group of refractive methods, ifthe resonance angle of the launched excitation light for generation ofthe surface plasmon resonance is taken as the quantity to be measured.Surface plasmon resonance can also be used for the amplification of aluminescence or the improvement of the signal-to-background ratios in aluminescence measurement. The conditions for generation of a surfaceplasmon resonance and the combination with luminescence measurements, aswell as with waveguiding structures, are described in the literature,for example in U.S. Pat. No. 5,478,755, No. 5,841,143, No. 5,006,716,and No. 4,649,280.

In this application, the term “luminescence” means the spontaneousemission of photons in the range from ultraviolet to infrared, afteroptical or other than optical excitation, such as electrical or chemicalor biochemical or thermal excitation. For example, chemiluminescence,bioluminescence, electroluminescence, and especially fluorescence andphosphorescence are included under the term “luminescence”.

In case of the refractive measurement methods, the change of theeffective refractive index resulting from molecular adsorption to ordesorption from the waveguide is used for analyte detection. This changeof the effective refractive index is determined, in case of gratingcoupler sensors, for example from changes of the coupling angle for thein- or outcoupling of light into or out of the grating coupler sensor,in case of interferometric sensors from changes of the phase differencebetween measurement light guided in a sensing branch and a referencingbranch of the interferometer. In case of a device for the generation ofsurface plasmon resonance, the change of the effective refractive indexcan be determined from a change of the resonance angle at which asurface plasmon (in a thin metal film deposited on a dielectricsubstrate) is generated. If a tunable excitation light source, both fora grating coupler sensor and for a device for generation of a surfaceplasmon resonance, a change of the effective refractive index can alsobe determined from a change of the excitation wavelength for satisfyingthe respective resonance condition, when the excitation light islaunched at a fixed angle close to the resonance angle.

Therefore, certain embodiments of a bioanalytical sensing platformaccording to the invention are characterized in that they are operapablefor analyte determination by means of the detection of a change of theeffective refractive index in the near field of the surface of saidsensing platform due to molecular adsorption on or desorption from saidsensing platform.

Specific embodiments of a bioanalytical sensing platform according tothe invention are operapable for analyte determination by means of thedetection of a change of the conditions for generation of a surfaceplasmon in a metal layer being part of said sensing platform, whereinsaid metal layer preferably comprises gold or silver. It is preferredthat said metal layer has a thickness between 40 nm and 200 nm, stillmore preferably between 40 nm and 100 nm.

The aforesaid refractive methods have the advantage, that they can beapplied without using additional marker molecules, so-called molecularlabels. The disadvantage of these label-free methods, however, is, thatthe achievable detection limits are limited to pico- to nanomolarconcentration ranges, dependent on the molecular weight of the analyte,due to lower selectivity of the measurement principle, which is notsufficient for many applications of modern trace analysis, for examplefor diagnostic applications.

Lower detection limits can be achieved, for example using methods basedon luminescence detection, especially if these methods are combined withoptical waveguide techniques, for example by fluorescence excitation inthe evanescent field of an optical waveguide.

Therefore, preferred embodiments of a bioanalytical sensing platformaccording to the invention are characterized in that said sensingplatform is operapable for analyte determination by means of thedetection of a change of one or more luminescences.

A bioanalytical sensing platform according to such an embodiment can beoperapable to receive excitation light in an epi-illuminationconfiguration.

It is preferred that the material of a bioanalytical sensing platformaccording to the invention, which material is in contact with themeasurement areas, is transparent, at least at one excitationwavelength, to a depth of at least 200 nm, measured from the surfacesupporting the immobilized biochemical or biological or syntheticrecognition elements in said measurement areas.

Characteristic for another embodiment of a bioanalytical sensingplatform according to the invention is, that it is operapable to receiveexcitation light in an transmission-illumination configuration.

In general, it is preferred that the materials of said sensing platformare transparent at least one excitation wavelength.

Characteristic for a preferred embodiment of a bioanalytical sensingplatform according to the invention is, that it is operapable as anoptical waveguide. It is further preferred that said optical waveguideis essentially planar.

For such an embodiment of a bioanalytical sensing platform operapable asan optical waveguide, it is preferred that it comprises an opticallytransparent (i.e. optically transparent at least one excitationwavelength) material selected from the group comprising silicates, suchas glass or quartz, thermoplastic or moldable plastics, such aspolycarbonates, polyimides, acrylates, especially polymethylmethacrylates, and polystyrenes.

It is especially preferred that a bioanalytical sensing platformaccording to the invention comprises an optical thin-film waveguide witha layer (a) being optically transparent at least one excitationwavelength on a layer (b) being optically transparent at least at thesame excitation wavelength, wherein the refractive index of layer (b) islower than the one of layer (a).

In order to couple excitation light into the wave guiding layer of abioanalytical sensing platform based on an optical waveguide, saidwaveguiding layer is in optical contact to at least one of the opticalcoupling elements selected from the group comprising prism couplers,evanescent couplers formed by joined optical waveguides with overlappingevanescent fields, distal end (front face) couplers with focusinglenses, preferably cylindrical lenses, located in front of a distal end(front face) of the waveguiding layer, and coupling gratings.

It is preferred that light incoupling into the optically transparentlayer (a) is performed by means of one or more grating structures (c)formed in layer (a).

It is further preferred that outcoupling of light guided in theoptically transparent layer (a) is performed by means of one or moregrating structures (c′) formed in layer (a), and wherein gratingstructures (c′) can have the same or different grating period asoptional additional grating structures (c).

Characteristic for one type of bioanalytical sensing platforms accordingto the invention, with coupling gratings (c) for incoupling ofexcitation light into the waveguiding layer (a) is, that an array of atleast 4 regions with at least two different “prefabricated patterns” aand b and, optionally, with one or more self-assembly systems (A, B, . .. ) deposited on the different “prefabricated patterns”, is locatedafter an incoupling grating (c), with respect to the direction ofpropagation of light guided in layer (a) after its incoupling by saidgrating.

Characteristic for another type of bioanalytical sensing platformsaccording to the invention, with coupling gratings (c) and/or (c′) is,that an array of at least 4 regions with at least two different“prefabricated patterns” a and b and, optionally, with one or moreself-assembly systems (A, B, . . . ) deposited on the different“prefabricated patterns”, is located on a coupling grating (c) or (c′).

For some applications it is preferred that a continuous coupling grating(c) or (c′) extends over at least 30% of the surface of said sensingplatform.

The optically transparent layer (b) should be characterized by lowabsorption and fluorescence, in the ideal case free of absorption andfluorescence. Additionally, the surface roughness should be low, becausethe surface roughness of the layer (b) does affect, dependent on thedeposition process to a more or less large extent, the surface roughnessof an additional layer (a) of higher refractive index, when it isdeposited on layer (a) as a waveguiding layer. An increased surfaceroughness at the boundary (interface) layers of layer (a) leads toincreased scattering losses of the guided light, which, however, isundesired. These requirements are fulfilled by numerous materials.

It is preferred that the material of the second optically transparentlayer (b) comprises an optically transparent material (i.e. opticallytransparent at least at one excitation wavelength) selected fro thegroup comprising silicates, such as glass or quartz, thermoplastic ormoldable plastics, such as polycarbonate, polyimides, acrylates,especially polymethylmethacrylates, and polystyrenes.

For a given layer thickness of the optically transparent layer (a), thesensitivity of an arrangement according to the invention increases alongwith an increase of the difference between the refractive index of layer(a) and the refractive indices of the adjacent media, i.e., along withan increase of the refractive index of layer (a). It is preferred, thatthe refractive index of the first optically transparent layer (a) ishigher than 1.8.

Another important requirement on the properties of layer (a) is, thatthe propagation losses of the light guided in layer (a) should be as lowas possible. It is preferred, that the first optically transparent layer(a) comprises a material selected from the group comprising TiO₂, ZnO,Nb₂O₅, Ta₂O₅, HfO₂, or ZrO₂, preferably especially from the groupcomprising TiO₂, Ta₂O₅, and Nb₂O₅. Combinations of several suchmaterials can also be used.

For a given material of layer (a) and given refractive index, thesensitivity does increase with decreasing layer thickness, up to acertain lower limiting value of the layer thickness. The lower limitingvalue is determined by the cut-off of light guiding, if the layerthickness falls below a threshold value determined by the wavelength ofthe light to be guided, and by an observable increase of the propagationlosses in very thin layers, with further decrease of their thickness. Itis preferred, that the thickness of the first optically transparentlayer (a) is between 40 and 300 nm, preferably between 70 and 200 nm.

If an autofluorescence of layer (b) cannot be excluded, especially if itcomprises a plastic such as polycarbonate, or for reducing the affect ofthe surface roughness of layer (b) on the light guiding in layer (a), itcan be advantageous, if an intermediate layer is deposited betweenlayers (a) and (b). Therefore, it is characteristic for anotherembodiment of the bioanalytical sensing platform according to theinvention, that an additional optically transparent layer (b′) withlower refractive index than and in contact with layer (a), and with athickness of 5 nm-10 000 nm, preferably of 10 nm-1000 nm, is locatedbetween the optically transparent layers (a) and (b).

It is preferred, that the grating structures (c) and optional additionalgrating structures (c′) have a period of 200 nm-1000 nm and a gratingmodulation depth of 3 nm-100 nm, preferably of 10 nm-30 nm.

Thereby it is preferred, that the ratio of the modulation depth to thethickness of the first optically transparent layer (a) is equal orsmaller than 0.2.

The grating structures can be provided in different forms (geometry). Itis preferred, that a grating structure (c) is a relief grating with anyprofile, such as right-angular, triangular or semi-circular profile, ora phase or volume grating with a periodic modulation of the refractiveindex in the essentially planar optically transparent layer (a).

Further embodiments of sensing platforms, which can be incorporated intoa bioanalytical sensing platform according to the invention if they areprovided with a “chemical surface pattern” as described above, as wellas methods for analyte determination performed with these sensingplatforms, are disclosed in U.S. Pat. Nos. 5,822,472, 5,959,292, andU.S. Pat. No. 6,078,705, and in the patent applications WO 96/35940, WO97/37211, WO 98/08077, WO 99/58963, PCT/EP 00/04869, and PCT/EP00/07529. Therefore, the embodiments disclosed therein are also part ofthis invention and incorporated by reference.

Another subject of the invention is a method for the simultaneousqualitative and/or quantitative determination of one or more analytes inone or more samples, wherein said samples are brought into contact withthe measurement areas on a bioanalytical sensing platform according tothe invention, and wherein the resulting changes of signals from saidmeasurement areas are measured.

For many applications methods are preferred wherein said changes ofsignals from the measurement areas are obtained upon using a label,which is selected from the group comprising luminescence labels,especially luminescent intercalators or “molecular beacons”, absorptionlabels, mass labels, especially metal colloids or plastic beads, spinlabels, such as ESR and NMR labels, and radioactive labels.

Other embodiments of a method for analyte determination according to theinvention are characterized in that analyte determination is performedupon detection of a change of the effective refractive index in the nearfield of the surface of said sensing platform due to molecularadsorption on or desorption from said sensing platform.

A special method is based on the detection of a change of the conditionsfor generation of a surface plasmon in a metal layer being part of saidsensing platform, wherein said metal layer preferably comprises gold orsilver.

For most applications, however, methods are preferred, wherein analytedetermination is performed upon detection of a change of one or moreluminescences.

The excitation light from one or more light sources can be launched onthe bioanalytical sensing platform in a configuration ofepi-illumination. In another embodiment of said method, excitation lightfrom one or more light sources is launched on the bioanalytical sensingplatform in a configuration of transmission-illumination.

Preferred are embodiments of a method for analyte determinationaccording to the invention, wherein the bioanalytical sensing platformcomprises an optical waveguide, which is preferably essentially planar,and wherein excitation light from one or more light sources is coupledinto said waveguide by means of an optical coupling element selectedfrom the group comprising prism couplers, evanescent couplers formed byjoined optical waveguides with overlapping evanescent fields, distal end(front face) couplers with focusing lenses, preferably cylindricallenses, located in front of a distal end (front face) of the waveguidinglayer, and coupling gratings.

Especially advantageous is an embodiment, wherein said bioanalyticalsensing platform comprises an optical thin-film waveguide, with a firstoptically transparent layer (a) on a second optically transparent layer(b) with lower refractive index than layer (a), wherein furthermoreexcitation light is incoupled into the optically transparent layer (a)by one or more grating structures formed in the optically transparentlayer (a), and directed, as a guided wave, to the measurement areaslocated thereon, and wherein furthermore the luminescence from moleculescapable to luminesce, which is generated in the evanescent field of saidguided wave, is detected by one or more detectors, and wherein theconcentration of one or more analytes is determined from the intensityof these luminescence signals.

In the method disclosed above, (1) the isotropically emittedluminescence or (2) luminescence that is incoupled into the opticallytransparent layer (a) and outcoupled by a grating structure (c) orluminescence comprising both parts (1) and (2) can be measuredsimultaneously.

For the generation of the luminescence or fluorescence in the methodaccording to the invention, a luminescence or fluorescence label can beused, that can be excited and emits at a wavelength between 300 nm and1100 nm.

The luminescence or fluorescence labels can be conventional luminescenceor fluorescence dyes or also so-called luminescent or fluorescentnano-particles based on semiconductors [W. C. W. Chan and S. Nie,“Quantum dot bioconjugates for ultrasensitive nonisotopic detection”,Science 281 (1998) 2016-2018].

The luminescence label can be bound to the analyte or, in a competitiveassay, to an analyte analogue or, in a multi-step assay, to one of thebinding partners of the immobilized biological or biochemical orsynthetic recognition elements or to the biological or biochemical orsynthetic recognition elements.

Additionally, a second or more luminescence labels of similar ordifferent excitation wavelength as the first luminescence label andsimilar or different emission wavelength can be used. Thereby, it isadvantageous, if the second or more luminescence labels can be excitedat the same wavelength as the first luminescence label, but emit atother wavelengths.

For other applications, it is advantageous, if the excitation andemission spectra of the applied luminescent dyes do not overlap oroverlap only partially.

In the method according to the invention, it can also be advantageous,if charge or optical energy transfer from a first luminescent dye actingas a donor to a second luminescent dye acting as an acceptor is used forthe detection of the analyte.

In addition, it can be of advantage, if besides determination of one ormore luminescences, changes of the effective refractive index on themeasurement areas are determined. It can be of further advantage, if theone or more luminescences and/or determinations of light signals at theexcitation wavelengths are performed polarization-selective. The methodallows also for measuring the one or more luminescences at apolarization that is different from the one of the excitation light.

The method according to the invention, according to any of theembodiments disclosed above, allows for the simultaneous or sequential,quantitative or qualitative determination of one or more analytes of thegroup comprising antibodies or antigens, receptors or ligands, chelatorsor “histidin-tag components”, oligonucleotides, DNA or RNA strands, DNAor RNA analogues, enzymes, enzyme cofactors or inhibitors, lectins andcarbohydrates.

The samples to be examined can be naturally occurring body fluids, suchas blood, serum, plasma, lymphe or urine, or egg yolk.

A sample to be examined can also be an optically turbid liquid orsurface water or soil or plant extract or bio- or process broths.

The samples to be examined can also be taken from biological tissue.

A further subject of the invention is the use of a bioanalytical sensingplatform and/or of a method for analyte determination, both according toany of the embodiments disclosed above, for quantitative or qualitativeanalysis for the determination of chemical, biochemical or biologicalanalytes in screening methods in pharmaceutical research, combinatorialchemistry, clinical and preclinical development, for real-time bindingstudies and the determination of kinetic parameters in affinityscreening and in research, for qualitative and quantitative analytedeterminations, especially for DNA- and RNA analytics, for thegeneration of toxicity studies and the determination of expressionprofiles and for the determination of antibodies, antigens, pathogens orbacteria in pharmaceutical product development and research, human andveterinary diagnostics, agrochemical product development and research,for patient stratification in pharmaceutical product development and forthe therapeutic drug selection, for the determination of pathogens,nocuous agents and germs, especially of salmonella, prions and bacteria,in food and environmental analytics.

In the area of biosensors, in particular in case of bioaffinity sensorsrelying on the specific detection of biologically relevant molecules ormolecular assemblies (e.g., DNA, RNA, proteins, cell receptors, etc.),patterning techniques are already playing a crucial role in the designof microarray sensor chips, which allow for more than one type ofanalyte to be analyzed on the same chip through controlled spatialarrangement of recognition units In particular, in microarray sensorchips with a total number of specific sensing areas (equivalent to“measurement areas” or “spots”) in the order of 10² to 10⁵ measurementareas per cm², the perfect spatial arrangement and localization ofactive recognition units (i.e. biological or biochemical or syntheticrecognition elements) in the measurement areas is mandatory for highquality (low probability of faulty measurements, high degree ofquantitativeness of the analytical measurement), reproducibleperformance. The stringent control over the spatial arrangement ofactive units becomes more important as the feature size and spacingdecreases. Such microarray sensors are generally fabricated by spatiallycontrolled immobilization of the biological or biochemical or syntheticrecognition elements on a chemically homogeneous chip surface usingtechniques such as ink jet printing, microdroplet capillary spotting,and others. Furthermore, there is a growing interest in cell-basedsensors, i.e. sensors where living cells are attached in a controlledfashion to chip surfaces and are used to sense environmental factors or,more specifically, cellular responses to their exposure to chemicals.

Critical issues for the bioaffinity and cell-based microarray sensorchips are

-   -   The precise location of the spots and their arrangement in a        regular geometric pattern    -   The uniformity of the spot size over the whole array    -   The preservation of the activity of the biological or        biochemical or synthetic recognition elements    -   The homogeneity of the distribution of the recognition units        within the spot area e.g. avoidance of “doughnut structures”),        and    -   The low signal (e.g. fluorescence) background of the area        immediately adjacent to the array spot.

The invented SMAP processes is able to provide solutions to a number offrequently observed specific problems and challenges that areencountered in practice and that are related to the above-mentionedcritical issues, for example:

-   -   The SMAP process is able to produce patterns with geometrically        organized areas of different wetability, e.g. areas of any        geometric form, size and interarray spacings (pitch) that are        hydrophilic in a “sea” or “background” that is hydrophobic. This        has two positive consequences: Firstly, independently of the        spotting technique used, the (aqueous) droplet, once it has        landed at the chip surface, is precisely located on the wettable        area since it “jumps into contact” due to the hydrophobic        surrounding area. Therefore, the precision of spot localization        can be higher than that of the spotting technique itself (which        is influenced by the mechanics of the system, drop formation,        size and detachment, electrostatic effects, etc.) and is        basically determined by the precision of the SMAP pattern. The        latter is extremely accurate. Secondly, the spot geometry after        drying of the spotted droplet can be precisely controlled, since        the contact area between droplet and chip surface is controlled        mostly through the size of the hydrophilic area; it can be        easily optimized for a given droplet volume.    -   Through the precise control of the droplet-chip interfacial        area, the surface-to-volume ratio of the landed droplet can be        precisely controlled. This is an important aspect, since it        allows a certain control over the evaporation rate (which        increases with increasing surface/volume ratio). Evaporation        rate is important if the process of immobilization of the        spotted recognition units takes some time, for example in case        where a chemical bond between the recognition unit and        functional chemical groups at the chip surface has to be formed        within the time of liquid-surface contact. Secondly, if both the        droplet volume (through the spotting technique) and the        droplet-surface contact area (through application of the        invented SMAP technique can be controlled, one can achieve        within the spotted area α much better control over the        homogeneity of the spatial distribution of the recognition units        adsorbed at the surface after evaporation of the droplet. If        this ratio is not well controlled, one often experiences        inhomogeneous distribution of the recognition unit, e.g. a        higher concentration in the center of the spot, or a “donut        shape” with higher concentrations at the border of a (e.g. round        spot). Such deviations from a perfectly homogeneous, controlled        size spot adversely affects both the adequate quantification of        the spot signal (e.g. fluorescence) and the maximum attainable        detection sensitivity.

For cell-based sensors, the precise placement of cells on geometricallywell-controlled, cell-adhesive spots is highly relevant [Chen C S,Mrksich M, Huang S, et al., Geometric control of cell life and death,Science 276 (5317): 1425-1428, May 30, 1997]. The SMAP technique allowsthe production of such well-controlled cell-adhesive patterns while atthe same time ensuring a very low tendency for cells to attach outsidethe adhesive areas (i.e., in the non-adhesive areas). Since thefunctionality of the cell is influenced by its morphology, a precisecontrol over the cell-surface contact area is a factor that is essentialfor the performance of the cell-based chip. Furthermore, the type anddensity of attachment sites for cells (e.g. peptides interacting withcell membrane receptors, focal contacts) are essential for both theattachment strength and cellular activity such as differentiation of thecell) [Rezania A, Healy K E, The effect of peptide surface density onmineralization of a matrix deposited by osteogenic cells, J Biomed MaterRes (4): 595-600, Dec. 15, 2000]. The SMAP technique is an idealtechnique for producing cell-adhesive patterns on cell-based sensorchips of high geometric fidelity, control over the surface density ofbiological functions interacting with the cell, and interfacialstability over time.

In terms of the chip technology and transducer requirements, the SMAPtechnique has the advantage of flexibility with respect to the choice ofthe appropriate substrate materials. The patterns can be produced onpreferably transparent substrates or chips, e.g. by using transparentmetal-oxide-based coatings on transparent substrates (glass, quartz,etc.). This allows one to use optical transmission technique for thecontrol of the patterns and for the use of optical detection techniquessuch as optical transmission (fluorescence) microscopy or opticalevanescent field technique (e.g. optical waveguide techniques).Alternatively, the SMAP technique can be applied to non-transparent,e.g. metallic reflecting chip surfaces. This may be advantageous ifdetection techniques requiring reflective surfaces such as reflectionmicroscopy or evanescent field techniques requiring metal surfacecoatings (e.g. surface plasmon resonance methods) are to be used.

There are a number of arguments why chemically patterned surfaces oftailored interactiveness with the environment are of interest toapplications in the area of biomaterials, biomedical devices andimplants.

Surfaces that are patterned into geometrically ordered areas that areadhesive to proteins and/or cells with a non-adhesive background willinteract with a biological medium in a more controlled and predictableway than is the case with homogeneous (unpatterned) or randomlyheterogeneous surfaces.

One argument for the exploitation of patterns in the size range of cells(few to few tens of micrometers) on biomaterials and implants is thefact that the size of cells (projection of cell shape on surface) can beinfluenced by the size (area) of the adhesive pattern. The size of thecell-surface contact area on the other hand has been shown to affect thedevelopment of the cells [Chen C S, Mrksich M, Huang S, et al.,Geometric control of cell life and death, Science 276 (5317): 1425-1428,May 30, 1997]. For example, proliferation and differentiation activityof osteoblastic cells react differently (mostly oppositely) to thedimensions of the cell-adhesive area and there is a particular size ofthe cell-surface contact area for which differentiation of osteoblastscells is fastest [Thomas C H, et al., J. Biomech. Eng. 121: 40, 1999;Thomas C H, et al. Proceedings of the Society for BiomaterialConference, Hawaii, 2000, p. 1222]. Furthermore, the form of the cellsand the formation of stress fibres can be tailored by choosingappropriate patterns of dimensions which contain both features withdimensions similar to those of cells (e.g. 5 to 100 μm) as well asconnected or disconnected features with features in the low micrometer(e.g. 1-5 μm) or submicrometer range, representative of subcellularfeatures such as membrane receptors or focal contacts. Since the stressfibres are important for cellular activity, not only the static behaviorbut also the dynamics of cells, e.g. motility, can be steered byappropriate patterns. In particular, anisotropic patterns may be used toeither direct cell motility along certain directions on the surface ofan implant or to impose anisotropy on the properties of the cellular ortissue interface that forms with time at the implant surface. Suchanisotropic properties at the interface may be beneficial to the shortand/or long-term performance of the biomaterial body or biomaterial-cellculture interface, for example in case of bone-related implants or intissue engineering of boneous material in vitro or in vivo (natural boneis a highly anisotropic material).

In a given situation in the body or in a primary cell culture, differentcells coexist and interact with the surface of the artificial material.It may therefore be of interest to the bioengineer to develop patternsthat have a positive influence on the behavior of different types ofcells at the surface. For example, in case of a bone implant, it may beadvantageous to have patterns that strongly support the attachment anddifferentiation of osteoblasts, but not of fibroblasts, in order tofavor the formation of a boneous, rather than fibrous, interfacialtissue. This may be achieved by choosing an optimum size and form of theadhesive pattern, an optimum distance between the features within thepattern and an optimum symmetry of the arrangement of the adhesive areaswithin features. Another form would be to choose an interactivebiological functionality within the adhesive pattern that interacts morestrongly with one type of cells that with other types. As an example, ithas been demonstrated that heparin-binding peptides of the type . . .KRSR . . . interact more strongly (almost selectively) with osteoblaststhan with fibroblast, while the integrin-binding peptide of type . . .RGD . . . interacts strongly with both types of cells [Hasenbein M E,Anderson T T, Bizios R, Proceedings of the Society for BiomaterialsConference, Hawaii, 2000, p.110; Dee K C, et al., Tissue Engineering 1(1995) 135; Dee K C, et al., J. Biomed. Mater. Res. 25 (1991) 771]. Onecould similarly envisage patterns that interact with cells that areimportant for healing, tissue integration and stability of implants,while such patterns do not support the attachment and proliferation ofbacteria.

Another application for chemically patterned surfaces is related to thecell type that is relevant in almost all in vivo implant applications,the macrophage. While macrophages fulfill an important function in“cleaning up” implantation sites and implant surfaces during the healingphase, their extended actions, in particular the occurrence offrustrated phagocytosis and formation from macrophages of multinucleargiant cells (“foreign body giant cells”, FBGC), may lead to sustainedinflammation and retarded or prevented healing reactions. Chemicallypatterned surfaces could improve the situation in at least two differentways: a) if the surface of an implant is patterned into cell-adhesiveand non-adhesive areas in dimensions significantly smaller than the sizeof an attached macrophage, the latter is expected to be prevented fromdeveloping a tight seal between the cell membrane and the surface. As aconsequence, the macrophage (and osteoclast)—typical excluded volumecannot form, which is a prerequisite for the sustained action ofgenerated, destructive acids, superoxides and peroxides within thisexcluded electrolyte volume. Therefore, an unfavorably massive degree ofchemical attack of biomaterials through macrophage activity could beprevented by using cell-adhesive/non-adhesive patterns of suitablegeometry. The same mechanism would hold for the action of osteoclasts ina bone environment. In a different approach (that can be combined withthe first one), pattern geometries can be designed that restrictmacrophage cells to individual sites at the surface, well separated fromeach other. In such a situation, unfavorable FBGC formation would besuppressed or at least reduced compared to a homogeneous or randomlyheterogeneous surface.

In summary, patterns may allow the biomedical engineer, interested indesigning implants or tissue engineering constructs with improvedperformance, to influence not only the type and density of cells at thebiomaterial-body or biomaterial tissue interface, but also on thedevelopment of cells at the interface with time, and therefore also onthe kinetics of formation and the properties of the resultinginterfacial tissue, which forms adjacent to the patterned biomaterial orimplant surface. While the chemical pattern is basicallytwo-dimensional, its effect in the biomaterial and tissue engineeringarea can be three-dimensional, exerting its influence also in the thirddimension, i.e. perpendicular to the surface, and up to distances muchlarger than the pattern dimensions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described below by providing information on thegeneral procedures of the selective molecular assembly patterningtechnique and detailed by way of specific examples and drawings asfollows:

FIG. 1. Regular, geometric (a) and random, statistically distributed (b)patterns with chemical composition A in a background of composition B.Surface view (a) and (b); cross section (c) without (top) and with(bottom) topographical change between A and B.

FIG. 2. Scheme of the technological steps in the fabrication of chemicalpatterns using the SMAP process. See text for discussion.

FIG. 3. Scheme for the production of patterned substrates by lithographyto be used in the context of the SMAP technique.

FIG. 4. Patterning of substrates in the submicron dimension range usingsmall particles such as nanosized polystyrene colloidal particles.

FIG. 5. Preparation of substrates for the SMAP process based on chemicalcontrast “written” with the help of a focused ion beam.

FIG. 6. Surface chemical composition of the prepatterned substratesurfaces (in cross section) used for the examples describing theapplication of the SMAP technique below.

FIG. 7. Fluorescence microscopy image of the SMAP treated surfaceshowing in dark gray the SiO₂ areas (5×5 μm) that are protein-resistantdue to the selective adsorption of PLL-g-PEG, while the light gray areasare selectively covered by the fluorescently labeled proteinstreptavidin that adsorbs to the TiO₂ areas which previously had beenhydrophobized by an alkane phosphate (DDP) self-assembled monolayer.

FIG. 8. Si-Wafer, coated with 90 nm TiO₂, and 12 nm SiO₂. Prepatternedsurface produced by dry etching of structures with dimensions 10×15 □m(central rectangle) and 2×15 □m lines. SMAP steps: 1) DDP, 2)PLL-g-PEG-biotin, 3) Albumin fluorescently labeled with Oregon Green, 4)Streptavidin fluorescently labeled with Texas Red (details are describedin the text). There is selective adsorption of the fluorescently labeledalbumin to the hydrophobic DDP areas, while fluorescently labeledstreptavidin binds to the biotinylated PLL-g-PEG, but not to thealbumin-passivated DDP areas.

FIG. 9. Adsorption of PLL-g-PEG-biotin to TiO₂ and SiO₂ surfacesrespectively, as a function of pH of the molecular assembly solution.The adsorbed mass of PLL-g-PEG-biotin was judged by quantitativefluorescence microscopy upon exposure of the PLL-g-PEG-biotin-treatedsurfaces to Oregon-Green-labeled streptavidin. The difference in pHdependence of the molecular adsorption process between the two surfacesforms the basis for the SMA patterning technique with contrast resultingfrom electrostatic interaction (“electrostatic contrast”, type II).

FIG. 10. Schematic drawing of SMAP according to type III, usinghydrophobic-hydrophilic contrast.

FIG. 11. List of selected combinations of prepatterning techniques withmolecular assembly processes. Standard micro- and nano-patterningmethodologies are listed on the left. Their objective is to produce aspecimen with two kinds of surfaces present, thus providing a materialcontrast for the SMAP patterning. Various examples of the latter arelisted on the right. DDP: dodecylphosphate or -phosphonate. Hb:hydrophobic anchoring group. X, Y—specific receptors (examples includebiotin, RGD-peptide, etc.).

DETAILED DESCRIPTION

The technological basis for the Selective Molecular Assembly Patterning(SMAP) is based on selective, spontaneous assembly out of solution ofmolecules with a physico-chemical, biochemical or biologicalfunctionality onto a substrate surface that contains a suitable patternprefabricated using any of the state-of-the-art surface-structuringtechniques. The chemical structure of the prefabricated substratepattern is chosen such that the subsequent molecular assembly stepselectively modifies one type of pattern, generally followed by a secondassembly process to coat the second type of pattern. Further selectivemodification steps may follow until the desired patterned surface orinterface architecture has been achieved.

General Flow Diagram for Creating Patterned Surfaces Based on SMAP

FIG. 2 schematically represents a typical sequence for the applicationof the SMAP technique (surface architecture shown in cross section):

-   (a) Substrate with homogeneous properties as the starting material.-   (b) Prepatterning of substrate into areas α and β with different    chemical composition (=generation of material contrast) using    state-of-the-art structuring/patterning techniques. It should be    noted, that this chemical patterning must not be necessarily be    associated with a topographical patterning, as shown in FIG. 2, but    can also be performed within the plane of the surface, e.g. using    local chemical modification upon exposure to laser light.-   (c) Application of a spontaneous molecular assembly system that    forms an adlayer selectively on area β (SMAP step A), but does not    (or to a much lower degree) interact with area α.    -   (d) Application of a spontaneous molecular assembly system that        forms an adlayer selectively on area α (SMAP step B).    -   (e) Depending on the system, one or several further        functionalization steps may be applied to complete the desired        surface or interface architecture.

Depending on the type of molecules and their degree of functionalproperties chosen in SMAP step A and B, the surface at stage (d) mayalready contain the final functionality needed for the givenapplication. Alternatively, the surface at stage (d) may containfunctional groups in areas A or B that can be converted (preserving thespatial selectivity) into the desired functionality in one or severaladditional modification steps (e).

Examples of Molecular Assembly Systems, Suitable for the SMAP Technique

Three types of molecular assembly processes are described as specificexamples that are suitable for use in the Selective Molecular AssemblyPatterning technique. They exploit a specific response to a particularset of physicochemical properties of the prepatterned substrate:

-   Type I: specific covalent or complex-coordinative binding, i.e.    “contrast based on selective chemical reactivity”.-   Type II: attractive versus repulsive electrostatic interactions,    i.e. “electrostatic contrast”.-   Type III: van der Waals interactions of hydrophobic molecular    segments with hydrophobic areas at the surface, i.e. exploiting    “hydrophobic-hydrophilic contrast”.    Type I: SMAP Using Alkane Phosphate Self-Assembled Monolayers    (“Selective Chemical Reactivity Contrast”):

Alkane phosphates and alkane phosphonates have been described in theliterature [M. Textor, L. Ruiz, R. Hofer, A. Rossi, K. Feldman, G.Hähner, N. D. Spencer, Structural Chemistry of Self-Assembled Monolayersof Octadecylphosphoric Acid on Tantalum Oxide Surfaces, Langmuir 16 (7):3257-3271 (2000)] to self-assemble on oxide surfaces such as tantalumoxide, titanium oxide, niobium oxide or aluminum oxide forming partiallyordered monolayers with well-defined physico-chemical properties. Theirapplication as homogenous (unpatterned) surfaces to the biomaterial andbiosensor field has been described in Swiss priority patent applicationNo. CH 1732/00. If aqueous solutions of alkane phosphates are used, ithas been observed that SAMs are formed on a variety of metal oxides suchas tantalum oxide, titanium oxide and niobium oxide, but NOT on siliconoxide. The silicon oxide surface remains uncoated. Therefore, if aprestructured substrate surface is used that contains, for example, apattern with silicon oxide patches and with titanium (or niobium ortantalum) oxide patches, only the titanium (or tantalum or niobium)oxide areas get coated with the alkane phosphate. If a methyl-terminatedalkane phosphate such as dodecyl phosphate (DDP) is used, a highcontrast in wetability results with hydrophobic areas corresponding toTiO₂-DDP, while the uncoated silicon oxide patches remain hydrophilic.In a second step, the silicon oxide patches may be coated with adifferent molecular assembly system, e.g. by adsorption ofprotein-resistant poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) onthe SiO₂ pattern, wherein g denotes the ratio between the number oflysine units and the number of poly(ethylene glycol) side chains, or byalkane phosphate with a different terminal functional group on the SiO₂pattern by adsorption from the corresponding organic solvents solution.

A corresponding example is given in Example 1 below.

Type II: SMAP Using Polyionic Polymers (“Electrostatic Contrast”)

Polyionic copolymers have been shown to assemble spontaneously oncharged surfaces forming stable adlayers due to electrostatic (and othertypes of) interactions if the charge of the polymer and of the surfaceare opposite (as described in patent application WO 00/65352). Forexample poly-L-lysine, which is positively charged at neutral pH,adsorbs to negatively charged surfaces—such as tissue-culturepolystyrene, or metal oxide surfaces such titanium oxide or siliconoxide. The use of polyethylene glycol grafted, polyionic copolymers isparticularly useful for the biosensor and biomaterial area, since theyform stable monolayers resistant to protein adsorption. This isimportant if the objective is to eliminate non-specific interactions ingeneral and impose specific interactions to certain areas of thepattern.

One typical way of exploiting the electrostatic contrast is to choose aprepatterned oxide surfaces based on two different oxides withsubstantially different isolelectric points (IEPs). By adjusting the pHof the molecular assembly solution such that the two types of patternarea are oppositely charged, conditions can be found for which thepolyionic polymer coats only one type of metal oxide, i.e. the one thatis oppositely charged in comparison to the polyionic polymer. In asecond step, the area that has not been coated in the first assemblystep serves as a substrate for the self-assembly of a different polymer,or of the same polymer with an additional functional group.

A corresponding example is given in Example 2 below.

Type III: SMAP Using Hydrophobic-Hydrophobic Interactions(“Hydrophobic-Hydrophilic Contrast”)

Functional copolymers that contain at least one segment that is highlyhydrophobic can be used within the SMAP technology if the prepatternedsurface contains hydrophobic and hydrophilic areas. Such copolymers willstrongly interact with the hydrophobic areas due to the hydrophobiceffect and via van der Waals (“hydrophobic-hydrophobic”) interactions.Although such polymers may also cover hydrophilic areas through weakerphysical interactions, their binding strength is likely to be weakenough to be removed by a solvent and suitable rinsing conditions,effectively resulting in a pattern that contains the polymer only in thehydrophobic areas. Copolymers of the “Pluronic” containing hydrophobicsegments composed of poly(propylene oxide) and hydrophilic segmentscomposed of poly(ethylene glycol) form a typical class of molecules thatare suitable for the SMAP technique in combination with prepatternedhydrophobic/hydrophilic surfaces. The PEG chains in the Pluronicsmolecules can be further functionalized with e.g. a biochemical orbiological functionality.

A corresponding example of the application of PEG-PPO-PEG within theSMAP technology is given in Example 3 below.

Prepatterned Substrate Fabrication

A variety of state-of-the-art techniques are principally suitable toprepattern substrates that are subsequently used in combination with thenovel SMAP technique. In particular, the following techniques can beused:

-   -   Photolithography using masks and photoresist coatings on        suitable substrates: standard lithography using visible, UV or        X-ray exposure, or more recently developed techniques such as        interference-based lithographic structuring [Rogers, J. A.,        Paul, K. E., Jackman, R. J., Whitesides, G. M. Generating ˜90 nm        features using near-field contact-mode photolithography with an        elastomeric phase mask. J. Vac. Sci. Technol. B16(1), (1998)        59-68.s]. A typical procedure using conventional lithography is        shown in FIG. 3.    -   Electron-beam lithography using masks and photoresist coatings        on suitable substrates (similar to FIG. 3, but with sequential        writing of the surface structures using an electron beam).    -   Lithographic techniques using colloids deposited onto surfaces,        schematically shown in FIG. 4 [Rogers, J. A., Paul, K. E.,        Jackman, R. J., Whitesides, G. M. Generating ˜90 nm features        using near-field contact-mode photolithography with an        elastomeric phase mask. J. Vac. Sci. Technol. B16(1), (1998)        59-68.s].    -   Focused ion beam in combination with a thin-film-deposited        substrate according the FIG. 5.

These techniques differ in terms of the range of feature sizes that canbe produced, parallel versus sequential “writing” of the patterns,costs, applicability to non-flat (e.g. curved) surfaces and requirementsfor the selection of suitable substrates. Depending on the envisagedsurface structure and application, a preferred technique from the listabove or any technique that allows one to chemically pattern surfacescan be chosen and applied to fabricate the prepatterned substrate to beused in the subsequent SMAP process.

Specific Examples of the SMAP Technique

Three specific examples are presented in the following. In terms of thefirst molecular assembly step (SMAP step A in FIG. 2), they are based onSMAP process of type I, II and III respectively. The substrate for theseexamples of SMAP-patterning has one of the structure shown in FIG. 6,where MeO stands for the appropriate transition metal oxide, such astitanium oxide, niobium oxide, tantalum oxide, or aluminum oxide, etc.,while SiO₂ stands for silicon oxide.

EXAMPLE 1 SMAP Based on Alkane Phosphate//Poly(L-lysine)-g-poly(ethyleneoxide) System (“Selective Chemical Reactivity Contrast”)

Out of aqueous solutions, dodecyl phosphate (DDP) self-assembles onmetal oxides but not on silicon oxide. Subsequent application ofPLL-g-PEG renders silicon oxide protein resistant, hence creating apattern of protein-adhesive and resistant areas. Protein adsorption tothe DDP-modified metal oxide surface is in this case non-specific anddue to hydrophobic interactions between the hydrophobic alkane phosphateSAM and hydrophobic moieties of the protein.

As a specific example, the following consecutive steps were applied:

-   -   a) The starting surface is produced using photolithography        according to general scheme in FIG. 3. A silicon wafer was first        coated with 100 nm TiO₂ followed by 10 nm SiO₂ using the        magnetron sputtering technique. After application of a        photoresist coating, irradiation through a corresponding mask,        dry etching through the SiO₂ layer using CF₄/CF₃H gas mixture        and removal of the photoresist, a pattern of 5×5 μm squares of        TiO₂ was produced while the rest of the surface remains SiO₂ (as        shown in FIG. 6 bottom).    -   b) The lithographically patterned TiO₂/SiO₂ surface is then        dipped in an aqueous solution of the ammonium salt of dodecyl        phosphoric acid (DDP, 0.5 mole/L) for 24 h at room temperature        (RT). A self-assembled monolayer of DDP forms on top of the TiO₂        5×5 □m areas, rendering these areas highly hydrophobic.    -   c) The surface is carefully rinsed using high purity water    -   d) The surface is exposed by dipping for 15 min into an aqueous        solution (in HEPES buffer) of poly(L-lysine)-g-poly(ethylene        glycol) (PLL-g-PEG; MW of PLL: 20,000 Da, g=3.5, MW of PEG:        2,000 Da; concentration of PLL-g-PEG=1 mg/mL; for details see:        [G. L. Kenausis, J. Vörös, D. L. Elbert, N. P. Huang, R.        Hofer, L. Ruiz, M. Textor, J. A. Hubbell, N. D. Spencer,        Poly(L-lysine)-g-poly(ethylene glycol) Layers on Metal Oxide        Surfaces: Attachment Mechanism and Effects of Polymer        Architecture on Resistance to Protein Adsorption, J. Phys. Chem.        B 104: 3298-3309 (2000)].    -   e) The surface is carefully rinsed using high purity water.    -   f) The surface is exposed by dipping to fluorescently (Texas        Red)-labeled streptavidin in HEPES buffer for 60 min at RT.    -   g) Final washing in HEPES buffer and in high purity H₂O.

FIG. 7 shows a fluorescence microscopy image of the SMAP treated surfaceshowing in dark gray to black the SiO₂ areas that are protein-resistantdue to the selective adsorption of PLL-g-PEG, while the light gray towhite areas are selectively covered by the protein streptavidin(fluorescently labeled) that adsorbs to the TiO₂ areas which previouslyhad been hydrophobized by an alkane phosphate self-assembled monolayer.

FIG. 8 demonstrates that the same SMAP process also works with morecomplex patterns.

This pattern has been produced in the following way: A silicon wafer wascoated by physical vapor deposition with 90 nm TiO₂, followed by 12 nmSiO₂. A photoresist was applied and a pattern with a central square of10×15 □m and lines of dimension 2×15 □m etched into the SiO2 layer. TheSMAP process consisted of the following sequential steps:

-   -   a) A hydrophobic dodecyl phosphate self-assembled monolayer        (DDP) was formed from aqueous solution (concentration: 0.5 mM,        RT) of the ammonium salt of DDP (dipping time: 24 h).    -   b) After rinsing, a PLL-g-PEG/PEG-biotin was deposited by        dipping the sample into an aqueous solution of        PLL-g-PEG/PEG-biotin (PLL-g-PEG; MW of PLL: 20,000 Da, g=3.5, MW        of PEG: 2,000 Da, MW of PEG-biotin: 3,200 Da; 50% of PEG chains        functionalized with biotin; concentration of        PLL-g-PEG/PEG-biotin=1 mg/mL).    -   c) The sample was exposed to an aqueous solution of        Oregon-Green-labeled albumin (concentration: 20 □g/mL; exposure        time: 1 h). Albumin covers selectively the hydrophobic areas,        i.e. the TiO₂-DDP pattern, but does not cover the        protein-resistant PLL-g-PEG/PEG-biotin areas.    -   d) Finally, the sample was exposed to a solution of        Texas-Red-labeled streptavidin (concentration: 1 mg/mL; exposure        time: 1 h) resulting in specific interactions with the biotin        functional groups of the PLL-g-PEG/PEG-biotin in the SiO₂        pattern areas, while the albumin passivated areas are resistant        to (further) protein adsorption.    -   e) The resulting strong chemical contrast shown in the        fluorescence microscopy image of FIG. 8 is due to albumin above        the TiO₂ areas and streptavidin above the SiO₂ areas.

EXAMPLE 2 SMAP Based on Poly(L-lysine)-g-poly(ethyleneglycol)//Poly(L-lysine)-g-poly(ethylene oxide-biotin) System(“Electrostatic Contrast”)

The difference in the isoelectric point between titanium oxide andsilicon oxide can be exploited to produce SMAP type II patterns based onelectrostatic contrast. This type of SMAP is illustrated using thespontaneous assembly of poly(L-lysine)-g-poly(ethylene glycol) atcharged surfaces, which is governed by electrostatic interactions. Thistechnique requires a starting surface with a pattern formed by twomaterials whose isoelectric points (IEP) are sufficiently different (IEPof SiO₂: ca. 2.5; IEP of TiO₂: ca. 6). FIG. 9 shows the dependence ofthe adsorbed mass of PLL-g-PEG(-biotin) (MW of PLL: 20,000 Da, g=3.5, MWof PEG: 2,000 Da) to SiO₂ and TiO₂ surfaces respectively, as a functionof the pH of the PLL-g-PEG aqueous solution to which the surfaces wereexposed for a time of 15 min. It is obvious from FIG. 9 that at pH=1.2,PLL-g-PEG (or functionalized PLL-g-PEG) can be selectively adsorbed tothe SiO₂ region, while the TiO₂ surface at the same pH remains uncovereddue to the repulsive interactions between the positively charged TiO2surface and the positively charged PLL-g-PEG.

The following protocol is a typical example of exploiting thedifferences in IEP and creating a pattern of areas that allow thearea-selective immobilization of streptavidin throughstreptavidin-biotin interactions. It involves the use the of a patternedTiO₂/SiO₂ substrate, followed by area-selective, spontaneous adsorptionof biotin-functionalized PLL-g-PEG (PLL-g-PEG/PEG-biotin) to the SiO₂pattern from aqueous solution at pH=1.2, followed by backfilling thebare oxide areas (particularly the TiO2 pattern) at a pH of 7 with(non-functionalized) PLL-g-PEG. The produced pattern can then be furthermodified by area-selective immobilization of streptavidin to thePLL-g-PEG/PEG-biotin areas. Such a surface can for example be used as asubstrate for the immobilization of biotinylated antibodies to thestreptavidin sites for application in protein sensing (proteomics) orfor the defined localization and attachment for cells in the area ofcell-based sensing.

Detailed SMAP Protocol:

-   -   Patterned SiO₂/TiO₂ surface is exposed to an aqueous solution of        PLL-g-PEG/PEG-biotin (concentration=1 mg/mL) at a pH of 1.2 and        at RT. Due to the surface charges at this pH, it only adsorbs to        the SiO₂ areas, while the TiO₂ areas, being strongly positively        charged, remain uncoated after this step.    -   Exposure of the surface to an aqueous solution of (unmodified)        PLL-g-PEG (concentration=1 mg/mL) at RT and at pH=7,        “backfilling” the TiO₂ areas and potentially present defects in        the PLL-g-PEG/PEG-biotin coating on SiO₂.

EXAMPLE 3 SMAP Based on PEG-PPO-PEG//Poly(L-lysine)-g-poly(ethyleneoxide-biotin) System (“hydrophobic-hydrophilic contrast”)

Example 3 relies on another type of contrast, namely the exploitation ofthe hydrophobic/hydrophilic contrast already described in Example 1. Ina further step, the hydrophobic areas are made protein- andcell-resistant (“non-interactive”) via the interaction with a triblockmolecule that contains both a hydrophobic block (to interact with thehydrophobic area of the pattern) and hydrophilic PEG blocks to renderthe adlayer protein-resistant. The other area of the pattern, thePLL-g-PEG/PEG-X, is cell-interactive due to X=specific peptidesinteracting with cell membrane receptors.

Protocol:

-   (a) MeO-DDP patches can be modified with PEG-based copolymers    possessing a hydrophobic backbone (PPO-PEG). This renders metal    oxide-DDP areas protein resistant. PLL-g-PEG/PEG-X (where X stands    for a specific receptor functionality, such as biotin or RGD    peptide) is used in the subsequent step to render silicon oxide able    to bind desired macromolecules, specifically and with high affinity.-   (b) The pattern of non-adhesive and specific areas can be inverted    by using a hydrophobic backbone-PEG copolymer bearing a functional    group.-   (c) A combination of hydrophobic backbone-PEG bearing one functional    group and PLL-g-PEG bearing another can be used, creating a pattern    of doubly-adhesive areas. The adhesion is in this case specific in    nature (as opposed to (a).

FIG. 10 illustrates schematically the SMAP according to type III, usinghydrophobic-hydrophilic contrast.

Overview of Further Examples Combining Different Prepatterning andSMAP-Based Techniques

FIG. 11 summarizes a selected, not exhaustive number of possibilities ofcombining prepatterning techniques to produce metal or oxide patternstogether with molecular assembly patterning to producebiologically-relevant chemical contrast using the SMAP technique.

Standard micro- and nano-patterning methodologies and recently developedtechniques such as colloidal lithography and interference patterning arelisted on the left. Their objective is to produce a specimen with twokinds of surfaces present, thus providing a material contrast for theSMAP patterning according to one of the three SMAP contrast methodsdiscussed above. Apart from the specific oxide patterns TiO₂/SiO₂, manyother oxide combinations are suitable for the SMAP process. Theirselection depends on the requirement of the SMAP process (i.e.coordinative interactions with alkane phosphates, surface charge forinteraction with polyionic copolymers, etc.) and of the application(e.g. requirement for optical transparency, etc.). Apart from SiO₂ andAl₂O₃, transition metal oxides often have the necessary properties to beused as at least one type of the prepattern material for later SMAPapplication. Also, metal surfaces, e.g. bulk metal specimens or metalfilms deposited onto suitable substrates can also be used as materialsfor prepatterning, since most of the metals are covered by an oxidefilm, Me_(x)O_(y), which can serve as one component for theprepatterning step.

In terms of the molecular assembly processes suitable to be combinedwithin the SMAP process, various examples are listed on the right ofFIG. 11. These represent a selection of preferred assembly techniques,but many other techniques are compatible with the SMAP process as longas they fulfill the requirements for one of the three types of SMAPprocesses.

Additionally, Table 1 compiles a more extensive list of molecularassembly techniques suitable for applications within the SMAP process.The table lists the type/class of molecules, their interaction withspecific examples of non-metal oxides, metal oxides or metals (with anatural or artificially produced oxide film at the surface) in terms ofthe binding or immobilization type, the non-interactiveness of thesurface following the assembly step (resistance to biomoleculeadsorption and cell attachment) or the non-specific interactivenesstowards biomolecules (e.g. proteins) and cells or the (bio)specificinteractiveness (either directly after the corresponding assemblyprocess or after an additional functionalization step).

Table 1 is also not exhaustive. Many more types of prepatternedsubstrate materials including metallic surfaces, nonmetallic, inorganicsurfaces (oxides, carbides, nitrides, etc.) or polymeric materials canbe used as long as they fulfill one or several of the requirements forapplications in the SMAP technology of type I, II or III (see above).

Similarly many more molecular assembly techniques than just the selectedexamples discussed above can be used in combination with suitableprepatterned surfaces, as long as they interact in a predictable waywith a particular type of prepatterned surface and react selectivelywith one type of the prepatterned areas and renders such a surfaceeither non-interactive, interactive in a non-specific way, orinteractive in a (bio)specific way. TABLE 1 Compilation of molecularassembly systems suitable for applications within the SMAP process(examples). The table lists the type/class of molecules, theirinteraction with specific examples of materials in terms of the bindingor immobilization type, the non-interactiveness of the surface followingthe assembly step (resistance to biomolecule adsorption and cellattachment) or the non-specific interactiveness towards biomolecules(e.g. proteins) and cells or the (bio)specific interactiveness (eitherdirectly after the corresponding assembly process or after an additionalfunctionalization step). Further Interaction Subsequent interactionbiochemical Molecule with substrate with or biological (type, example)(type, examples) biological medium functionalization Alkane phosphatesAdsorbs a) Non-specific — or onto: transition adsorption of b)Modification phosphonates metal oxides protein(s) through (CH₃— suchthrough hydrophobic- specific terminated), as oxides hydrophobicinteractions. interaction e.g. CH₃—(CH₂)_(x)—PO₄ of Ti, Nb, b) Protein-and with functionalized with Zr, Ta; cell-resistant albumin x = 2-24other metal surface after surface, oxides that passivation with e.g.between form metal- albumin or functionalized streptavidin phosphatealbumin, and complexes e.g. biotinylated biotinylated such as albuminalbumin aluminum c) Protein- and c) Modification oxide. cell-resistantthrough Does NOT surface after specific adsorb onto adsorption ofinteraction silicon oxide di- or multi- with functionalized blockpolymer PEG-PPO. with hydrophobic segments and hydrophilic, non-interactive segments, e.g. PEG- PPO (‘Pluronics’, see Example 3). ThePEG chains can be further functionalized, e.g. with biotin. Oligo- orAdsorbs Resistant to — poly(ethylene onto: transition protein adsorptionoxide)- metal oxides and cell modified alkane such attachment phosphatesas oxides or of Ti, Nb, phosphonates, Zr, Ta; e.g.(EO)_(y)—(CH₂)_(x)—PO₃ other metal with oxides that x = 2-24, y = 2-50form metal- phosphate complexes such as aluminum oxide. Does NOT adsorbonto silicon oxide Oligo- or Adsorbs Resistant to Biospecificpoly(ethylene onto: transition protein adsorption attachment oxide)-metal oxides and cell of antigen, modified alkane such attachment e.g.streptavidin phosphates as oxides to or of Ti, Nb, biotin orphosphonates, Zr, Ta; of cells with terminal other metal through(□-positioned) oxides that specific biological form metal- peptide-ligand, e.g. phosphate cell membrane biotin or peptide complexesinteractions. such as aluminum oxide. Does NOT adsorb onto silicon oxideOligo- or Adsorbs Resistant to Biological poly(ethylene onto: transitionprotein adsorption moiety can oxide)- metal oxides and cell be attachedmodified alkane such attachment to functional phosphates as oxides groupor of Ti, Nb, through covalent phosphonates, Zr, Ta; bond, e.g., withterminal other metal peptide, (□-positioned) oxides that protein,reactive form metal- enzyme. chemical phosphate group, e.g. N- complexeshydroxysuccinimidyl, such as maleimide, vinylsulfone, aluminum oxide.Does NOT adsorb onto silicon oxide Polycationic. Adsorbs to Protein- and— PEG-modified oxide surfaces cell-resistant copolymers, with surfacee.g. PLL-g-PEG negative (see Examples surface 1 and 2) charge, i.e. at asolution pH that is higher than the isoelectric point of the oxidesurface, e.g. at pH > 5 for TiO₂ or at pH > 1.5 for SiO₂. Does NOT (orless) adsorb to positively charged surfaces. Polycationic. Adsorbs toProtein- Interacts PEG-modified oxide surfaces resistant. specificallycopolymers, with with e.g. PLL-g-PEG negative cells (if (see Examplessurface ligand = specific 1 and 2), with charge, peptide), part or allof i.e. at a or with DNA the PEG chains solution pH or RNA iffunctionalized that is ligand is with a bioactive higher than anoligonucleotide ligand the or isoelectric with streptavidin point of ifthe oxide ligand = biotin. surface, e.g. at pH > 5 for TiO₂ or at pH >1.5 for SiO₂. Does NOT (or less) adsorb to positively charged surfaces.Polyanionic Adsorbs to Protein- and — PEG-modified oxide surfacescell-resistant copolymers, with e.g. positive Poly (glycolic surfaceacid)-g-PEG charge, (see Examples i.e. at a 1 and 2) solution pH that islower than the isoelectric point of the oxide surface, e.g. at pH > 5for TiO₂ or at pH > 1.5 for SiO₂. Does NOT (or less) adsorb tonegatively charged surfaces. Polyanionic Adsorbs to Protein- InteractsPEG-modified oxide surfaces resistant. specifically copolymers, withwith e.g. PLL-g-PEG positive cells (if (see Examples surface ligand =specific 1 and 2), with charge, peptide), part or all of i.e. at a orwith DNA the PEG chains solution pH or RNA if functionalized that isligand is with a bioactive lower than an oligonucleotide ligand the orisoelectric with streptavidin point of if the oxide ligand = biotin.surface, e.g. at pH > 5 for TiO₂ or at pH > 1.5 for SiO₂. Does NOT (orless) adsorb to negatively charged surfaces. Polyanionic or Adsorbs toPotein-resistant Biological polycationic oxide surfaces moiety canPEG-modified with be attached copolymers positive to functional (seeExamples surface group 1 and 2), with charge, through covalent part orall of i.e. at a bond, e.g., the PEG chains solution pH peptide,functionalized that is protein, with a reactive lower than enzyme.functional the group isoelectric point of the oxide surface, e.g. atpH > 5 for TiO₂ or at pH > 1.5 for SiO₂. Does NOT (or less) adsorb tonegatively charged surfaces.

After application of the SMAP process, depending on the type of theprocess and the molecular assembly system used, the surface layer in oneof the two (or more) patterns may already contain a biospecific functionfor interaction with biomolecules or cells or it may contain a suitablereactive (functional) group that allows one to attach biospecificfunctions. Examples are given in Table. 1

1. Device with chemical surface patterns with biochemical or biologicalrelevance on substrates with prepatterns of at least two different typesof regions (α, β, . . . ), whereas at least two different, consecutivelyapplied molecular self-assembly systems (A, B, . . . ) are used in a waythat at least one of the applied assembly systems (A or B or . . . ) isspecific to one type of the prefabricated patterns (α or β or . . . ).2. Device according to claim 1 where the specificity is achieved throughself-assembly of alkane phosphates or alkane phosphonates from aqueoussolutions (assembly system A) in combination with prepatterned surfaceswhereas only one type of the prepattern area (α) forms a molecularlyassembled layer A of alkane phosphates, while the other prepatternarea(s) (β, . . . ) remains uncoated.
 3. Device according claim 2 whereα is an oxide, nitride or carbide of a metal that chemically interactswith phosphates and/or phosphonates, in particular transition metaloxides such as titanium oxide, tantalum oxide, niobium oxide, zirconiumoxide, or non-transition metal oxides that chemically interact withphosphates or phosphonates, and where β is an oxide that does notinteract, in particular silicon oxide.
 4. Device according to claim 1where the specificity is achieved through assembly of polyionic,PEG-grafted polymers (B) from aqueous solution at a pH chosen such thatone of the two or more prepattern areas (β) is charged oppositely incomparison to the polyionic copolymer and becomes coated by thecopolymer due to electrostatic interactions, while the other prepatternarea(s) (α) at the same pH carries a charge of same sign as thecopolymer and does not or does less become coated.
 5. Device accordingto claim 4 where the prepattern area β is an oxide, nitride or carbidewith an isoelectric point (IEP) that is lower than that of area α andthe assembly system is a (at the pH of application) polycationiccopolymer and the pH of the assembly system solution is chosen betweenthe IEP of area α and area β.
 6. Device according to claim 4 where theprepattern area β is an oxide, nitride or carbide with an isoelectricpoint (IEP) that is higher than that of area α and the assembly systemis a (at the pH of application) polyanionic copolymer and the pH of theassembly system solution is chosen between the IEP of area α and area β.7. Device according to claim 1 where the specificity is achieved throughself-assembly of a di- or multiblock copolymer with hydrophobic andhydrophilic segments interacting with a substrate where one of theprepattern area (α) is more hydrophobic than the remaining areas, andtherefore gets coated by the di- or multiblock copolymer A while theother prepattern area (β) remains uncoated or less coated.
 8. Deviceaccording to claim 7 where the di- or multiblock copolymer is apolypropylene oxide (PPO)-poly(ethylene glycol) (PEG) copolymerimparting protein resistance to the more hydrophobic surface.
 9. Deviceaccording to claim 7 where the hydrophobic prepattern area (α) iscomposed of a hydrophobic polymer or of an oxide that has beenhydrophobized through silanization or application of an alkane phosphateself-assembly system, while the hydrophilic prepattern area is eithercomposed of a hydrophilic polymer or is an inherently hydrophilic oxideor is an oxide that has been made permanently hydrophilic throughapplication of a self-assembled monolayer using a molecule withhydrophilic terminal functional group.
 10. Device according to claim 2where in a second molecular assembly step B the prepattern area β thathas not been coated with the alkane phosphate becomes coated with aprotein-resistant polymeric layer, leading to a final pattern that isinteractive with a biological environment (proteins, cells) in areas Aand not interactive (protein- and cell-resistant) in areas B.
 11. Deviceaccording to claim 10 where B is the assembly of a polyionic PEG coatedcopolymer, adsorbing onto the oppositely charged area β, e.g.polycationic poly(L-lysine)-g-poly(ethylene oxide) adsorbing at pH ofbetween 2 and 8 onto negatively charged silicon oxide.
 12. Deviceaccording to claim 4 where in a second step the prepattern area αbecomes coated with a functionalized polyionic PEG-grafted copolymer Athrough application of the second self-assembly solution at a pHdifferent from step 1, at which pH the area α is now oppositely chargedin comparison to the polyionic copolymer A and becomes coated with thefunctionalized polymer, leading to a final pattern that is interactivewith a biological environment (proteins, cells) in areas A andnon-interactive in areas B.
 13. Device according to claim 12 where thepolyionic PEG-grated copolymer is functionalized at the end of the PEGchains through covalent linkage to a biologically active group such asbiotin interacting with streptavidin, or a peptide or a protein,interacting specifically with receptors in cell membranes.
 14. Deviceaccording to claim 7 where in a second step the more hydrophilic area(β) that has not been coated in assembly step A gets coated in thesecond assembly step B with a molecule that induces specific ornon-specific interaction with the biological environment.
 15. Deviceaccording to claim 7 where B is a functionalized polyionic PEG-graftedcopolymer according to claim 13 that interacts electrostatically withthe oppositely charged surface β or is an alkane phosphate that turnsthe area β into a hydrophobic, non-specifically interactive arearesulting in a final interactive/non-interactive pattern.
 16. Deviceaccording to claim 13 where a oligo(ethylene oxide) functionalizedalkane phosphate is used as the molecular assembly system A, leading toa non-interactive area A, while the area β are subsequently treated withan assembly system B that renders this area interactive, e.g. byadsorbing a functionalized, polyionic PEG-grafted copolymer.
 17. Deviceaccording to claim 8 where a functionalized (e.g. biotin or peptide orreactive chemical group attached at end of PEG chains) PPO-PEG diblockor PEG-PPO-PEG triblock, or multiblock copolymer is used to render thecorrespondingly covered area specifically interactive, followed by asecond assembly system that renders the remaining area non-interactive,e.g. through adsorption of a polyionic PEG-coated copolymer.
 18. Deviceaccording to claim 1 where after application of assembly system A and Bthe resulting interactive/non-interactive pattern is further modifiedthrough selective treatment of area A and/or B with biochemically orbiologically relevant molecules.
 19. Device according to claim 18 wherethe selective treatment is a nonspecific adsorption of proteins or otherbiomolecules to the area that is (non-specifically) interactive, e.g.hydrophobic or a selective interaction with ligands previouslyimmobilized in step A or B, e.g. streptavidin interacting specificallywith biotin ligand on one of the pattern area.
 20. Device according toclaim 19 where living cells are added to patterned surfaces and becomeimmobilized selectively on one of the pattern area, through interactionwith selectively and nonspecifically adsorbed protein or proteins, orthrough specific interactions with bioligands such as peptides orproteins that have in a previous step been immobilized through covalentattachment to one of the pattern areas.
 21. A bioanalytical sensingplatform comprising a device according to claim 1 and at least onebiological or biochemical or synthetic recognition element, for thespecific recognition and/or binding of one or more analytes and/or forthe specific interaction with said analyte(s), immobilized eitherdirectly or mediated by a self-assembled layer and/or by anadhesion-promoting layer on at least one of the different types ofregions a or b or . . . .
 22. A bioanalytical sensing platform accordingto claim 21, wherein the biological or biochemical or syntheticrecognition element is attached to at least one of the appliedself-assembly systems A or B, or adsorbs on at least one of saidself-assembly systems.
 23. A bioanalytical sensing platform according toclaim 22, wherein the biological or biochemical or synthetic recognitionelements are immobilized in a one-or two-dimensional array of discretemeasurement areas, wherein a single discrete measurement area is definedby the area occupied by said immobilized biological or biochemical orsynthetic recognition elements on an individual, closed region a or b.24. A bioanalytical sensing platform according to claim 23, wherein upto 1,000,000 measurement areas are provided in a two-dimensionalarrangement on one device with chemical surface pattern, and wherein asingle measurement area occupies an area between 10⁻⁴ mm² and 10 mm².25. A bioanalytical sensing platform according to claim 24, wherein themeasurement areas are arranged at a density of at least 10, preferablyof at least 100, most preferably of at least 1000 measurement areas persquare centimeter.
 26. A bioanalytical sensing platform according toclaim 25, wherein the biological or biochemical or synthetic recognitionelements are selected from the group comprising proteins, such as mono-or polyclonal antibodies or antibody fragments, peptides, enzymes,aptamers, synthetic peptide structures, glycopeptides, oligosaccharides,lectins, antigens for antibodies (e.g. biotin for streptavidin),proteins functionalized with additional binding sites, nucleic acids(such as DNA, RNA, oligonucleotides or polynucleotides) and nucleic acidanalogues (such as peptide nucleic acids, PNA) or their derivatives withartificial bases, soluble, membrane-bound proteins, such asmembrane-bound receptors and their ligands.
 27. A bioanalytical sensingplatform according to claim 26, wherein whole cells or cell fragmentsare immobilized for specific recognition and detection of one or moreanalytes.
 28. A bioanalytical sensing platform according to claim 27,wherein whole cells or cell fragments are immobilized in discretemeasurement areas.
 29. A bioanalytical sensing platform according toclaim 28, wherein less than 100, preferably less than 10, mostpreferably only 1-3 cells or cell fragments are immobilized permeasurement area.
 30. A bioanalytical sensing platform according toclaim 29, which works for analyte determination by means of a label,which is selected from the group comprising luminescence labels,especially luminescent intercalators or molecular beacons, absorptionlabels, mass labels, especially metal colloids or plastic beads, spinlabels, such as ESR and NMR labels, and radioactive labels.
 31. Abioanalytical sensing platform according to claim 29, which isoperapable for analyte determination by means of the detection of achange of the effective refractive index in the near field of thesurface of said sensing platform due to molecular adsorption on ordesorption from said sensing platform.
 32. A bioanalytical sensingplatform according to claim 29, which is operapable for analytedetermination by means of the detection of a change of the conditionsfor generation of a surface plasmon in a metal layer being part of saidsensing platform, wherein said metal layer preferably comprises gold orsilver.
 33. A bioanalytical sensing platform according to claim 29,which is operapable for analyte determination by means of the detectionof a change of one or more luminescences.
 34. A bioanalytical sensingplatform according to claim 33, which is operapable to receiveexcitation light in an epi-illumination configuration.
 35. Abioanalytical sensing platform according to claim 34, wherein thematerial of said sensing platform, which is in contact with themeasurement areas, is transparent, at least one excitation wavelength,to a depth of at least 200 nm, measured from the surface supporting theimmobilized biochemical or biological or synthetic recognition elementsin said measurement areas.
 36. A bioanalytical sensing platformaccording to claim 33, which is operapable to receive excitation lightin an transmission-illumination configuration.
 37. A bioanalyticalsensing platform according to claim 36, wherein the materials of saidsensing platform are transparent at least one excitation wavelength. 38.A bioanalytical sensing platform according to claim 37, which isoperable as an optical waveguide.
 39. A bioanalytical sensing platformaccording to claim 38, characterized in that it is an essentially planarwaveguide.
 40. A bioanalytical sensing platform according to claim 38,characterized in that it comprises an optically transparent materialselected from the group comprising silicates, such as glass or quartz,thermoplastic or moldable plastics, such as polycarbonates, polyimides,acrylates, especially polymethyl methacrylates, and polystyrenes.
 41. Abioanalytical sensing platform according to claim 40, characterized inthat it comprises an optical thin-film waveguide with a layer (a) beingoptically transparent at least one excitation wavelength on a layer (b)being optically transparent at least at the same excitation wavelength,wherein the refractive index of layer (b) is lower than the one of layer(a).
 42. A bioanalytical sensing platform according to claim 41, whereinthe waveguiding layer of said platform is in optical contact to at leastone of the optical coupling elements selected from the group comprisingprism couplers, evanescent couplers formed by joined optical waveguideswith overlapping evanescent fields, distal end (front face) couplerswith focusing lenses, preferably cylindrical lenses, located in front ofa distal end (front face) of the waveguiding layer, and couplinggratings.
 43. A bioanalytical sensing platform according to claim 42,wherein incoupling into the optically transparent layer (a) is performedby means of one or more grating structures (c) formed in layer (a). 44.A bioanalytical sensing platform according to claim 42, whereinoutcoupling of light guided in the optically transparent layer (a) isperformed by means of one or more grating structures (c′) formed inlayer (a), and wherein grating structures (c′) can have the same ordifferent grating period as optional additional grating structures (c).45. A bioanalytical sensing platform according to claim 44, wherein anarray of at least 4 regions with at least two different prefabricatedpatterns a and b according to claim 1 and, optionally, with one or moreself-assembly systems (A, B, . . . ) deposited on the differentprefabricated patterns, is located after an incoupling grating (c), withrespect to the direction of propagation of light guided in layer (a)after its incoupling by said grating.
 46. A bioanalytical sensingplatform according to claim 44, wherein an array of at least 4 regionswith at least two different prefabricated patterns a and b according toclaim 1 and, optionally, with one or more self-assembly systems (A, B, .. . ) deposited on the different prefabricated patterns, is located on acoupling grating (c) or (c′).
 47. A bioanalytical sensing platformaccording to claim 46, wherein a continuous coupling grating (c) or (c′)extends over at least 30% of the surface of said sensing platform.
 48. Abioanalytical sensing platform according to claim 47, wherein anadditional, at least at one excitation wavelength optically transparent,layer (b′) with lower refractive index than and in contact with layer(a), and with a thickness of 5 nm-10 000 nm, preferably of 10 nm-1000nm, is located between the optically between the optically transparentlayers (a) and (b).
 49. A bioanalytical sensing platform according toclaim 48, wherein layer (b) comprises an optically transparent (i.e.optically transparent at least one excitation wavelength) materialselected from the group comprising silicates, such as glass or quartz,thermoplastic or moldable plastics, such as polycarbonates, polyimides,acrylates, especially polymethyl methacrylates, and polystyrenes.
 50. Abioanalytical sensing platform according to claim 49, wherein therefractive index of layer (a) is higher than 1.8.
 51. A bioanalyticalsensing platform according to claim 50, wherein layer (a) comprises amaterial selected from the group comprising TiO₂, ZnO, Ta₂O₅, HfO₂, andZrO₂, preferably especially from the group comprising TiO₂, Ta₂O₅, andNb₂O₅.
 52. A bioanalytical sensing platform according to claim 51,wherein the thickness of layer (a) is between 40 and 300 nm, preferablybetween 70 and 200 nm.
 53. A bioanalytical sensing platform according toclaim 52, wherein gratings (c) or (c′) have a period of 200 nm-1000 nmand a modulation depth of 3 nm-100 nm, preferably of 10 nm-30 nm.
 54. Amethod for the simultaneous qualitative and/or quantitativedetermination of one or more analytes in one or more samples, whereinsaid samples are brought into contact with the measurement areas on abioanalytical sensing platform according to claim 21, and wherein theresulting changes of signals from said measurement areas are measured.55. A method according to claim 54, wherein said changes of signals fromthe measurement areas are obtained upon using a label, which is selectedfrom the group comprising luminescence labels, especially luminescentintercalators or molecular beacons, absorption labels, mass labels,especially metal colloids or plastic beads, spin labels, such as ESR andNMR labels, and radioactive labels.
 56. A method according to claim 54,wherein analyte determination is performed upon detection of a change ofthe effective refractive index in the near field of the surface of saidsensing platform due to molecular adsorption on or desorption from saidsensing platform.
 57. A method according to claim 54, wherein analytedetermination is performed upon detection of a change of the conditionsfor generation of a surface plasmon in a metal layer being part of saidsensing platform, wherein said metal layer preferably comprises gold orsilver.
 58. A method according to claim 54, wherein analytedetermination is performed upon detection of a change of one or moreluminescences.
 59. A method according to claim 58, wherein excitationlight from one or more light sources is launched on the bioanalyticalsensing platform in a configuration of epi-illumination.
 60. A methodaccording to claim 58, wherein excitation light from one or more lightsources is launched on the bioanalytical sensing platform in aconfiguration of transmission-illumination.
 61. A method according toclaim 58, wherein the bioanalytical sensing platform comprises anoptical waveguide, which is preferably essentially planar, and whereinexcitation light from one or more light sources is coupled into saidwaveguide by means of an optical coupling element selected from thegroup comprising prism couplers, evanescent couplers formed by joinedoptical waveguides with overlapping evanescent fields, distal end (frontface) couplers with focusing lenses, preferably cylindrical lenses,located in front of a distal end (front face) of the waveguiding layer,and coupling gratings.
 62. A method according to claim 61, wherein saidbioanalytical sensing platform comprises an optical thin-film waveguide,with a first optically transparent layer (a) on a second opticallytransparent layer (b) with lower refractive index than layer (a),wherein furthermore excitation light is incoupled into the opticallytransparent layer (a) by one or more grating structures formed in theoptically transparent layer (a), and directed, as a guided wave, to themeasurement areas located thereon, and wherein furthermore theluminescence from molecules capable to luminesce, which is generated inthe evanescent field of said guided wave, is detected by one or moredetectors, and wherein the concentration of one or more analytes isdetermined from the intensity of these luminescence signals.
 63. Amethod according to claim 62, wherein (1) the isotropically emittedluminescence or (2) luminescence that is incoupled into the opticallytransparent layer (a) and outcoupled by a grating structure (c) or (c′)or luminescence comprising both parts (1) and (2) is measuredsimultaneously.
 64. A method according to claim 63, wherein, for thegeneration of said luminescence, a luminescent dye or a luminescentnano-particle is used as a luminescence label, which can be excited andemits at a wavelength between 300 nm and 1100 nm.
 65. A method accordingto claim 64, for the simultaneous or sequential, quantitative orqualitative determination of one or more analytes of the groupcomprising wherein antibodies or antigens, receptors or ligands,chelators or histidin-tag components, oligonucleotides, DNA or RNAstrands, DNA or RNA analogues, enzymes, enzyme cofactors or inhibitors,lectins and carbohydrates.
 66. A method according to claim 64, whereinthe samples to be examined are naturally occurring body fluids, such asblood, serum, plasma, lymphe or urine or egg yolk or optically turbidliquids or surface water or soil or plant extracts or bio- or processbroths or are taken from biological tissue.
 67. The use of abioanalytical sensing platform according to claim 21 and/or of a methodaccording to claim 54 for quantitative or qualitative analysis for thedetermination of chemical, biochemical or biological analytes inscreening methods in pharmaceutical research, combinatorial chemistry,clinical and preclinical development, for real-time binding studies andthe determination of kinetic parameters in affinity screening and inresearch, for qualitative and quantitative analyte determinations,especially for DNA- and RNA analytics, for the generation of toxicitystudies and the determination of expression profiles and for thedetermination of antibodies, antigens, pathogens or bacteria inpharmaceutical product development and research, human and veterinarydiagnostics, agrochemical product development and research, for patientstratification in pharmaceutical product development and for thetherapeutic drug selection, for the determination of pathogens, nocuousagents and germs, especially of salmonella, prions and bacteria, in foodand environmental analytics.
 68. Device according to claim 1 comprisinga biomedical device with patterns in the size range of cells, typically5 to 100 micrometer, interconnected or not, isotropic or anisotropic, toinfluence or control cell form and attachment area, cell morphology,cytoskeleton organization, cell proliferation, cell differentiation andthe expression of factors within the cell and to the extracellularmatrix.
 69. A biomedical device according to claim 68, where cells areosteogeneic precursor cells, osteoblasts, osteoclasts, fibroblasts,smooth muscle cells, endothelial cells, epithelial cells, nerve cells,macrophages.
 70. Device according to claim 1 comprising a biomedicaldevice with patterns of size below 5 micrometer and above 10 nanometer,which are representative of subcellular features such as membranereceptors or focal contacts in order to influence the formation ofstress fibres, the organization of the cytoskeleton and the migration ofthe cell at the surface.
 71. A biomedical device fabricated according toclaims 68 or 70 with cell-adhesive patterns that contain specificligands such as peptides, proteins and antibodies and that are used tointeract more specifically with one kind of cells than with others withthe aim to influence the formation of assembly of preferred cell typesand the formation of a preferred type of tissue at the implant/bodyinterface.
 72. A biomedical device fabricated according to claims 68 or70 with cell-adhesive patterns that contain specific ligands such aspeptides and that are used to interact specifically with one or aselected number of cell membrane receptors, e.g. of the integrinreceptor or heparin-type receptor type.
 73. Patterns according to claim71, whereby the peptides contain one or several of the following aminoacid sequences: RGD, KRSR, YIGSG, FHRRIKA, DGEA, CSRARKQAASIKVAVSADR,MAPLRPLLIL, ALLAWVALAD, QESCKGRCTE, GFNVDKKCQC, DELCSYYQSC, CTDYTAECKP,QVTRGDVFTM, PEDEYTVYDD, GEEKNNATVH, EQVGGPSLTS, DLQAQSKGNP, EQTPVLKPEE,EAPAPEVGAS, KPEGIDSRPE, TLHPGRPQPP, AEEELCSGKP, FDAFTDLKNG, SLFAFRGQYC,YELDEKAVRP, GYPKLIRDVW, GIEGPIDAAF, TRINCQGKTY, LFKGSQYWRF, EDGVLDPDYP,RNISDGFDGI, PDNVDAALAL, PAHSYSGRER, VYFFKGKQYW, EYQFQHQPSQ, EECEGSSLSA,VFEHFAMMQR, DSWEDIFELL, FWGRTSAGTR, QPQFISRDWH, GVPGQVDAAM, AGRIYISGMA,PRPSLAKKQR, FRHRNRKGYR, SQRGHSRGRN, QNSRRPSRAT WLSLFSSEES, NLGANNYDDY,RMDWLVPATC, EPIQSVFFFS, GDKYYRVNLR, TRRVDTVDPP, YPRSIAQYWL, GCPAPGHL,MRIAVICFCL, LGITCAIPVK, QADSGSSEEK, QLYNKYPDAV, ATWLNPDPSQ, KQNLLAPQTL,PSKSNESHDH, MDDMDDEDDD, DHVDSQDSID, SNDSDDVDDT, DDSHQSDESH, HSDESDELVT,DFPTDLPATE, VFTPVVPTVD, TYDGRGDSVV, YGLRSKSKKF, RRPDIQYPDA, TDEDITSHME,SEELNGAYKA, IPVAQDLNAP, SDWDSRGKDS, YETSQLDDQS, AETHSHKQSR, LYKRKANDES,NEHSDVIDSQ, ELSKVSREFH, SHEFHSHEDM, LVVDPKSKEE, DKHLKFRISH, ELDSASSEVN,MKTALILLSI, LGMACAFSMK, NLHRRVKIED, SEENGVFKYR, PRYYLYKHAY, FYPHLKRFPV,QGSSDSSEEN, GDDSSEEEEE, EEETSNEGEN, NEESNEDEDS, EAENTTLSAT, TLGYGEDATP,GTGYTGLAAI, QLPKKAGDIT, NKATKEKESD, EEEEEEEEGN, ENEESEAEVD, ENEQGINGTS,TNSTEAENGN, GSSGGDNGEE, GEEESVTGAN, AEGTTETGGQ, GKGTSKTTTS, PNGGFEPTTP,PQVYRTTSPP, FGKTTTVEYE, GEYEYTGVNE, YDNGYEIYES, ENGEPRGDNY, RAYEDEYSYF,KGQGYDGYDG, QNYYHHQ, STGSKQRSQN, RSKTPKNQEA, SNVILKKRYN, MVVRACQCH. 74.Biomedical device according to claim 68, where the size of the adhesivesites are chosen such that macrophages can adsorb to such patterns, butnot nucleate into polynuclear cells of the Foreign Body Giant Cell(FBGC) type.
 75. Biomedical device according to claim 68 where thepatterns are applied to three-dimensional objects.
 76. Biomedical deviceaccording to claim 75, where the objects are products or components ofproducts such as catheters, stents, dental and maxillofacial implants,osteosynthesis plates or screws, artificial joint components, spinesurgery device such as cages, vascular and cardiovascular devices suchas heart valves or audiological devices, all of which are used incontact with a biological environment in a living body (“in vivo”), suchas body fluid, blood, biological tissue.
 77. Biomedical device accordingto claim 68, which is used as a substrate in cell culture testing (“invitro”) to influence and organize cell attachment to such substrate. 78.Biomedical device according to claim 68, whereby the substrate is madeout of a metal or alloy, a polymer, a ceramic material or a compositematerial.
 79. Endoprosthesis and implant according to claim 78 used injoint replacement (hip, knee, ankle, shoulder, elbow, wrist, finger,etc.) and bone fracture fixation (plates, screws, pins, nails, etc.)respectively where their whole or selected parts of their contactsurface area with hard or soft tissue respectively is patterned by SMAP.These endoprostheses and implants, i.e. the substrate for application ofSMAP may consist of metal, polymer or ceramic materials as well as ofcombinations of these materials types, i.e. composites. Suchendoprostheses and implants are intended to be used in humans andanimals, alone or in combination with any additional auxiliary materialslike bone cement, bioactive or bioinductive substances.